1. Field of the Invention
The present invention relates to crystallography and, more particularly, to the pre-filling of microplates with a precipitating solution for transport and handling prior to utilization.
2. Description of the Prior Art
Crystallography is an extremely useful tool for scientists, and is therefore a field of research attracting a lot of interest. It is a powerful means that provides precise and detailed description of the three-dimensional structure of the molecules, and is of great help in the understanding of their functions. Crystallography of macromolecules like proteins is extensively used today, academically as well as industrially.
Although three-dimensional structures of simple proteins have been obtained through crystallographic methods that necessitate crystal formation, it is not always easy to obtain crystals from macromolecules. For example, the preferred conditions for the crystallization of a given molecule can take several hundreds if not thousands of trials. As a result, means and methods have been developed to perform a great number of trials relatively quickly, including hanging-drop and sitting-drop methods. All such methods use the benefit of vapor diffusion to obtain the crystals.
According to the vapor diffusion technique, a small volume of a macromolecule sample is mixed with an approximately equal volume of crystallization or precipitating solution. The resulting drop of liquid is sealed in a chamber with a much larger reservoir volume of crystallization solution. The drop is kept separate from the reservoir of crystallization solution either by hanging the drop from a crystallization surface or by sitting the drop on a pedestal above the level of the crystallization solution in the reservoir. Over time, the crystallization drop and the crystallization solution equilibrate via vapor diffusion of volatile chemical species. Equilibration by vapor diffusion occurs between the drop and the reservoir until supersaturation of the macromolecule is achieved, resulting in crystallization of the macromolecule sample in the drop.
The process of growing biological macromolecule crystals remains, however, a highly empirical process during which different crystallization parameters are varied using a trial and error approach. Usually those parameters are: pH, temperature, and salt concentration in the crystallization drop, the concentration of the macromolecule to be crystallized and the concentration of the precipitation agents (of which there are hundreds). Using a trial and error approach, one would like to try as many crystallization conditions (screening of crystallization conditions), by varying parameters described above, to have a better probability to obtain the crystallization condition that will allow the growth of well diffracting macromolecule crystals. To allow laboratory to work faster by not having to prepare all the different solutions necessary to perform many different trials, company like Hampton Research have introduced pre-formulated screens usually composed of multiples of 24 tubes of 10 mL of different crystallization solutions. Following the venue of Hampton Research on the market other company like MDL, Emerald Biostructures and Jena Biosciences started introducing pre-formulated crystallization screens. One problem with all those crystallization solutions is that they need to be transferred into the crystallization plates either manually or automatically when performing crystallization trials. This represents a long and arduous work that has to be performed by qualified and skilled technical personnel.
Furthermore, the transfer of the crystallization solutions into the microplates generally needs to be done rapidly in order to avoid evaporation of the crystallization liquid. Such evaporation would alter the composition of the solution, thereby creating problem to reproduce the same condition if crystal growth occur. Typically, transfer is done using a micropipette. The technicians have to open a tube containing the solution and then pipette the solution into the crystallization reservoir. To make transfer faster in SBS standard crystallization plates, one can use multi-channel micro-pipette with SBS standard deep well block which are pre-filled with up to 2 mL times 96 different crystallization conditions (to set-up 96 wells crystallization plates). To do so, researchers need to centrifuge the block (to prevent cross-contamination of the wells when removing the seal), unseal the block and start to transfer the solutions. One problem is that evaporation start to occur as soon as the block is unsealed; it is an important issue since the whole procedure can take several minutes. Another problem is that the block needs to be resealed if researcher do not used all the solutions. To use all the solution, researchers need to pre-fill a minimum of 10 plates even if they typically need up to 3 plates (1 per temperature). It is also important to mention that errors with labeling and dispensing, when handling hundreds of different solutions, and cross-contamination can occur.
Another approach is to use sophisticated automated liquid handling stations developed by companies like Tecan, Gilson or Robodesign. Those stations are useful when performing routine molecular biology experiments involving DNA manipulation since the number of solutions to handle is reduced and also evaporation is less an issue (proteins are far more unstable over time and temperature than DNA). For example, U.S. Pat. No. 6,148,878 issued on Nov. 21, 2002 to Ganz et al. discloses an automated machine for filling a plurality of microplates. One problem with this system is that it is not suitable for pre-filling multiple microplates to do crystallization trials since there is no provision of any means that could be used for automatically and efficiently sealing the wells of the microplates for storage purposes before utilization thereof.
In summary, filling of the wells of a microplate with a crystallization solution is an arduous task which if not well executed under strict pre-established criteria might produce an undesirable wide variation in experimental results while jeopardizing the repeatability of the trials.
Therefore, efforts have been made to find ways for pre-filling the plates. One known approach involves sealing the top of a crystallization microplate with tape or a heat sealed foil. Following storage and transportation, the seal needs to be removed prior to setup crystallization experiments to provide access to the top surface of the plate where grease is typically applied about each well. When removing the seal, there is a risk to take away liquid that might be present on the seal due to transportation of the plate or condensation when temperature change occurs, thus creating unwanted experimental variations by changing the volume or the concentration of the solution left in the reservoir. To remove the liquid on the sealing mean, researchers need to centrifuge the plate prior to use; this can be complicated to automate and also requires extra equipments and an extra step. Furthermore, the sealing of the top of the microplate and the centrifugation solution is only applicable to hanging-drop crystallization plates. Indeed, in the case of a sitting-drop plate where the crystallization surface is located within the well below the top surface of the plate and the top sealing means, the crystallization solution contained in the well is free to contact the crystallization surface, thereby contaminating the same. Furthermore, in this case, centrifugation cannot be used to remove liquid from the undersurface of the top sealing means since such centrifugation will cause portion of the crystallization solution to contact the crystallization surface which is even more problematic.
According to applicants' knowledge, no one has heretofore been able to pre-fill, prior to transport/manipulation, plates or microplates used to carry sitting-drop crystallization experiments. This is due to the fact that the crystallization surface (where the drop containing the macromolecule to be crystallized sits) will most likely be spoiled/contaminated by the precipitating solution during manipulation/transport between the places were the microplates are filled and where the crystallization drop set-up is done.
Researchers are always trying to use smaller drops when carrying vapor diffusion crystallization experiments in order to minimize the amount of protein used during crystallization. When using small drops of less than 1 mL some problems may occur because equilibration can be to fast to allow good crystallization conditions to be reached. There is thus a need to find some means to slow down or control the equilibration process when conducting vapor diffusion experiments with very small volume of solution. Various approaches have been tested to control/modify/change the time required for full equilibration between the drop and the mother-liquid (the crystallization solution). Some have tried to use oil to slow down/control the evaporation process (D'Arcy et al. (1996) J. Crystal Growth 168, 175-180). One drawback of this approach resides in the fact that an extra step needs to be done to dispense oil in the reservoir over the crystallization solution. Moreover, when preparing a hanging-drop crystallization set-up, the oil can possibly reduce the quality of the image observed under the microscope. Finally, in some cases, the wells of the microplates can be too small to add enough oil.
In summary the process of growing biological macromolecule crystals remains a highly empirical process. Testing numerous conditions of variables that affect crystal growth, by means of thousands of crystallization trials, eventually leads to the optimal crystallization condition. Consequently the market is in need of inventions to help to rapidly and easily generate many reproducible crystallization trials. Therefore, there is a need for a device that will ease the setup of multiple crystallization trials while minimizing the risk of errors and cross-contamination during such setup. Another important need is to have a device/method that would increase experimental reproducibility due to evaporation or liquid losses between the filling of the microplates with the crystallization solutions and the setup of the crystallization drops. Finally, there is a need for a device that would also allow researchers to control the rate of vapor-diffusion equilibrium of multiple crystallization trials in multiple microplates.